This is a competing renewal application whose overall objective is to define the mechanisms of interferon (IFN)-signaling in malignant cells. IFNs exhibit important antineoplastic properties in vitro and in vivo and are key elements in the immune surveillance against cancer, but the mechanisms by which they generate such effects remain to be defined. We have provided the first evidence that the mTOR signaling cascade is engaged by IFN receptors (IFNRs) and regulates cap-dependent mRNA translation via control of the eukaryotic initiation factor 4E (eIF4E) and the eIF4F complex. Remarkably, our studies have provided evidence for signaling specificity and differential use of mTORC2 complexes by IFNs, as compared to oncogenic signals. Our studies suggest dual regulatory roles for mTORC2 complexes in IFN-signaling, controlling downstream pathways that regulate both transcription and mRNA translation of ISGs. The current proposal is a systematic approach to dissect the functions and roles of these complexes in IFN-signaling and to define their relevance in the generation of the antineoplastic effects of IFNs. Specific aim 1 will identify upstream IFNR-generated signals that lead to mTORC2 activation and will determine the mechanisms of specificity of mTORC2 engagement by the Type I IFNR. It includes studies to dissect the role of IFNR-associated kinases in the process; studies to define whether differential engagement of distinct Sin1 isoforms accounts for specificity in the IFN-system; and screening efforts to identif novel Rictor- and Sin1-interacting IFN-signaling elements. Specif ic aim 2 will define the mechanisms by which mTORC2 complexes control ISG expression and their roles in the generation of IFN-inhibitory responses in malignant cells. It includes experiments to define the roles of mTORC2 complexes in IFNR-activated signaling cascades that regulate transcriptional activation and mRNA translation of ISGs; experiments on the effects of IFN-activated mTORC2 complexes on AGC kinases; and studies to dissect the requirement of distinct downstream effector elements of mTOR pathways in the generation of IFN-antiproliferative responses. Specif ic aim 3 will examine the roles of mTOR-dependent signals in the antineoplastic effects of IFNs in Ph (-) myeloproliferative neoplasms (MPNs). JAK2V617F mouse models will be established in AKT -/-, S6K -/- and Pdcd4 -/- KO mice, and the ability of IFN to induce antileukemic responses in vivo, in the presence or absence of distinct effectors of the pathway, will be assessed. The activation of IFN-dependent mTOR pathways in primary hematopoietic precursors from patients with MPNs in vitro and in vivo will be assessed and correlated with IFN-sensitivity. Altogether, these studies will advance our understanding of the signaling mechanisms controlling generation of IFN-antitumor responses and will provide important information on the events that lead to malignant cell resistance to IFNs. Ultimately, they may form the basis for new approaches to overcome IFN-resistance.